Mammalian cells employ a hypoxic response pathway to sense and respond to changes in oxygen availability. This pathway contributes to both the physiological adaptation to hypoxic stress as well as the progression of a number of disease states in which oxygen supply is compromised, including ischemia and cancer. The Hypoxia Inducible transcription Factor (HIF) lies at the heart of this pathway and mediates expression of several targets relevant to tumorigenesis including genes involved in glycolysis, cell survival/proliferation, angiogenesis, and invasion/metastasis. Recently, our laboratory and others have shown that both HIF stability and coactivator recruitment are acutely regulated by oxygen-dependent prolyl and asparaginyl hydroxylases, respectively. While HIF can be so induced as a function of intratumoral hypoxia, HIF activity is frequently upregulated in cancers by a variety of oxygen-independent mechanisms resulting from genetic alterations that activate oncogenes or inactivate tumor suppressors. The objectives of this proposal are twofold. (1) We will investigate a relationship we've identified between HIF activity and a candidate tumor suppressor gene, Inhibitor of Growth family member 4 (ING4), reported to repress angiogenesis and tumor growth in glioblastomas. Proposed in this Aim is a combination approaches for the study of (a) ING4-mediated repression of HIF (b) the mechanism by which ING4 is recruited to HIF via the HIF prolyl hydroxylase (c) the identification of ING4-associated factors recruited to HIF and perhaps other transcription factors and (d) the relationship between ING4 expression, HIF association, and HIF activity in human tumors. (2) HIF is composed of two subunits, each of which contains two PAS domains whose roles in HIF function have not yet been fully elucidated. Using insights derived from the recently determined solution structures of the HIF PAS-B domains as a starting point we will (a) investigate the structure/function relationships of the HIF PAS domains via in vitro assays and cell culture models (b) demonstrate the potential therapeutic utility of disrupting HIF function through alterations to the PAS domains both in cell culture and in mouse tumor models and (c) pursue the development of small molecule HIF antagonists that directly bind the PAS domains. Together, these studies will increase our understanding of HIF regulation and may provide new avenues for therapeutic intervention.